Cloning of β-1,3-glucanase gene from Trichoderma virid LTR-2 and its ex-pression in Pichia pastoris

Abstract:

Text We designed a pair of primers according to fungal glucanase genes obtained from GenBank and cloned a novel β-1,3-glucanase gene (glu) from Trichoderma virid LTR-2 cDNA by PCR. Then we linked the fragment with pMD18-T vector and sequenced it. The sequence analysis indicated that glu was composed of 2289 nucleotide residues. The fragment contained an Open Reading Frame coding 762 amino acids and was similar to previous reports. Translated amino acids sequence of glu contained two Conservative Districts of β-1,3-glucanase which were RVVYIPPGTY and AASQNKVAYF. By nucleotide blasting in NCBI glu showed high homology to three β-1,3-glucanase genes from Trichoderma sp., especially with T.harzianum bgn3.1 and Hypocrea virens bgn13.1, which the homology reached 93%. The sequence was submitted to GenBank and the Ac-cession Number is EF176582. Then we connected glu gene with the Pichia pastoris shuttle vector-pPIC9K. The recombinant plasmid named pGLU14 was transformed into methylotropic yeast P. pastoris KM71 after linearization. The recombinant strain KGLU14 expressing β-1,3-glucanase at high level was obtained through plate screening. The SDS-PAGE result indicated that molecular weight of the recombinant β-1,3-glucanase was about 80kDa and the activity of the recombinant enzyme could reach 889U/mL in liquid culture.